Study On The Expression And Function Of Genes Involved In The Embryonic Cardiovascular Development Of Zebrafish

The cardiovascular system is one of the earliest organ systems to develop and function in the animal embryos,which is responsible for transporting oxygen,nutrients,and white blood cells to the cells in all parts of the body.Therefore the cardiovascular development and cardiovascular diseases have always been a scientific issue of concern.There is no doubt that a thorough study of the molecular mechanism of cardiovascular development will be more helpful to reveal the pathogenesis of cardiovascular diseases and to develop effective treatment methods.The cardiovascular development is precisely regulated by a very complex gene regulation network,in which there are more gene functions need to be investigated.Now zebrafish has becoming more popular model animal for the research of cardiovascular development and cardiovascular disease.The fish has many advantages over other model animals.In the present study,the RNA sequencing was conducted on zebrafish embryos at three key stages of the cardiovascular development,i.e.,18,24,and 48 hours post fertilization(hpf).Genes related to cardiovascular development was screened out from the differentially expressed genes(DEGs)of different development stages and their functions were studied.In addition,the roles of the long noncoding RNAs during the embryonic cardiovascular development of zebrafish was also investigated.The experimental results are listed as follows:(1)Transcriptome sequencing on the zebrafish embryos at three critical stages of cardiovascular developmentIn order to screen out the genes related to the cardiovascular development,three critical stages of the cardiovascular development of zebrafish embryos were selected,i.e.,18(T1),24(T2),and 48 hpf(T3),and transcriptome analysis was performed using the RNA-seq.The sequencing analysis results showed that 2605 DEGs were screened between T1-T2,among which 2003 genes were up-regulated and 602 genes were down-regulated.Total 3275 DEGs were screened from T2-T3,among which2420 genes were up-regulated and 855 genes were down-regulated.A total of 6446 DEGs were screened from T1-T3,among which 4608 genes were up-regulated and1838 genes were down-regulated.Total 644 common DEGs was gained using taking intersection of DEGs of the three periods,including 568 up-regulated genes,76down-regulated genes.The result of KEGG pathway analysis showed that 644 DEGs,a total of 42 genes enriched in the two pathways,including circulation system and cardiovascular disease.At the same time,167 genes of |log FC|>5 was screened out from the three periods,among which 6 genes were down-regulated and 161 genes were up-regulated.Through KEGG pathway analysis of 167 genes,11 genes enriched in the circulation system and cardiovascular disease pathways were screened out and were regarded as the key genes.Finally,using the RNA-seq and analysis,four protein-coding genes desma,cntn2,lama2 and ryr2 b related to the embryonic cardiovascular development in zebrafish were obteined for the next funciton study.(2)Functional study on the genes related to the embryonic cardiovascular development of zebrafishTo further investigate the function of the protein-coding genes(desma,cntn2,lama2 and ryr2b)related to cardiovascular development,the loss of function was performed using CRISPR-CAS9(clustered regularly interspaced short palindromic repeats-CAS9).The expression of several cardiovascular marker genes was detected in the loss-of-function embryos using the whole-mount embryo in situ hybridization in order to characterize their effects on the cardiovascular development.The marker genes included the cardiomyocyte-specific marker gene cmlc2,the vascular endothelial-specific marker gene fli1,and lateral plate mesoderm transcription factors hand2,and the somite mesoderm-specific marker myo D.The results indicated that the desma deficient mutant F0 generation was with 9 bases missing,lama2 with 6 bases missing,and cntn2 with 4 bases missing,respectively,by using the knockout method of CRISPR-CAS9.F1 generation carrying desma-mutation and lama2-mutaton was obtained,respectively by cross-fertilizing by wild adult and the loss-of-function F0 mutants,and the sequencing results of 24 hpf embryo of F1 generation proved that the mutation was successfully inherited to F1 generation.The F0 generation of ryr2b-knockout was confirmed to be successful by the 24 hpf embryo sequencing.In the process of mutation screening,the function of these genes was also studied.The expression of four markers related to cardiovascular development was detected by whole-mount in situ hybridization.The function of these genes was evaluated by analyzing the changes of cardiovascular phenotype.The test results are as follows: 1)The expression of cmlc2 in desma loss-of-function mutant embryos at 24 hpf was reduced,which may affect the size of cardiac chambers at subsequent development period and thus affecting cardiac function.The expression of fil1 in 48 hpf embryos indicated that the intersegmental blood vessels had sprout defects,suggesting that the loss of desma function also affected the development of blood vessels.Hand2 expression test showed that abnormal expression in the branchial arch in the mutant embryos was found.The changes of somite morphology in the mutant embryos were found by Myo D expression,which also affected the development of intersegmental blood vessels to a certain extent.2)Cmlc2 expression detection revealed that the abnormal heart loop was found in the cntn2 mutant embryos.Normal hearts loops to right in all vertebrates,while left-looped in the cntn2 mutant embryos.Abnormal looping may lead to congenital defects in the inflow and outflow tract.The result showed that the expression of hand2 was significantly decreased in the 5th branchial arch compared with the control,Myo D expression in mutant embryos was reduced.3)Fli1 expression showed that abnormal intersegmental vessel sprout in lama2–deficient mutant was found.There were no obvious differences in the expression of hand2 and myo D between lama2 loss of function embryos and control embryos.4)Cmlc2 expression was reduced in ryr2 b loss-of-function mutant embryos compared with the control embryos.This result indicated that ryr2 b loss of function affected the development of embryonic cardiomyocytes.Ventricle and atrium of the heart tube in the 34 hpf embryo were elongated and linear,which would affect the function of the heart.The expression of the other three genes fli1,hand2 and myo D was not significantly changed.(3)Spatiotemporal expression and function of long non-coding RNAs in the embryonic cardiovascular development of zebrafishLnc?H007,lcn?H001 and TCONS?00028652 were found and enriched in the heart in the previous report.This study first investaged the spatiotemporal expression of these lnc RNAs in the embryos of zebrafish.Their relationship with the adjacent genes was analyzed using biological databases and the bioinformatics research was carried out.Then,their spatiotemporal expression in the developmental embryos were detected by q PCR and whole-mount embryo in situ hybridization technology.The results of bioinformatics analysis showed that TCONS?00028652 was a long intergenic non-coding RNA(linc RNA),lnc?H001 was intron type,and lnc?H007 was antisense type.The q PCR results showed that the three long non-coding RNAs were expressed at different developmental stages of the embryo,and were also expressed in all the detected adult tissues.The expression of TCONS?00028652 was higher in the brain and liver than in other tissues.The expression of lnc?H001 was higher in the brain,while the expression of lnc?H007 was higher in the heart than in other tissues.This result suggests that these lnc RNAs may be involved in neural development of zebrafihs embyros and tissues repairment of adult.In order to study TCONS?00028652 function,the knock-down mutants were generated by injecting the morpholino targeted TCONS?00028652 to 1?2 cell of transgenic zebrafish [Tg(fil1:EGFP)].The cardiovascular development can be visualized by the transgenic fish with fli1 fluorescent protein.The expression of fli1 in knock-down embryos further verified the changes of vascular phenotype using whole-mount embryo in situ hybridization.The result showed that the sprout defects of intersegmental vessels were observed in both transgenic fish and 48 hpf embryos hybridized by digoxigenin-labeled antisense probe of fil1,which fully proved that the knock-down of TCONS?00028652 affected the cardiovascular development of zebrafish embryos.In order to further verify the function of TCONS?00028652 in the cardiovascular development,the loss of function of TCONS?00028652 was performed using CRISPR-CAS9.The expression of cmlc2 in the mutant embryos was then examined by whole-mount in situ hybridization.The result showed that the heart of 48 hpf and72 hpf embryos of the gene-knockout mutants had a serious defect in heart morphology,with some embryos heart developing linear tube and atrial no well-separated from ventricular.This result suggests that loss of function of the lnc RNA TCONS?00028652 might affect the cardiovascular development and cardiac morphogenesis of zebrafish embryos.The main conclusion of this study:(1)Transcriptome sequencing of zebrafish embryos at three critical stages of cardiovascular development was performed and 42 genes involved in the cardiovascular development were identified.(2)This study successfully obtained the F0 generation positive mutants of 4genes by gene-knockout method CRISPR-CAS9,and proved that the mutations of desma and lama2 could be inherited to the F1 generation.The 4 genes were characterized as the cardiovascular-development-related genes.The loss of function mutants of the 4 genes generated in this study may provide an important basis for further function study.(3)Long non-coding RNAs lnc?H007,lnc?H001,and TCONS?00028652 were expressed at different developmental stages of the embryo,and were also expressed in the heart and other tissues of adult fish.The function of TCONS?00028652 was studied by morpholino knockdown method and the gene-knockout method CRISPR-Cas9,respectively,and the results revealed that TCONS?00028652 was involved in the cardiovascular development and morphogenesis of the zebrafish heart.